Raju R V, Anderson J W, Datla R S, Sharma R K
Department of Pathology, College of Medicine, Royal University Hospital, University of Saskatchewan, Saskatoon, Canada.
Arch Biochem Biophys. 1997 Dec 1;348(1):134-42. doi: 10.1006/abbi.1997.0333.
Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. We have isolated full-length cDNA encoding bovine spleen NMT (sNMT). The single long open reading frame of 1248 bp of sNMT specifies a protein of 416 amino acids with a predicted mass of 46,686 Da. The protein coding sequence was expressed in Escherichia coli resulting in the production of functionally active 50-kDa NMT. Deletion mutagenesis showed that the C-terminus is essential for activity whereas up to 52 amino acids can be deleted from the N-terminus without affecting the function. One of the N-terminal deletions resulted in threefold higher NMT activity. Genomic Southern analysis indicated the presence of two strong hybridizing bands with three different restriction enzyme digests suggesting the possibility of two copies of the NMT gene in the bovine genome. RNA blot hybridization analysis of total cellular RNA prepared from bovine brain, heart, spleen, lung, liver, kidney, and skeletal muscle probed with bovine sNMT cDNA revealed a single 1.7-kb mRNA. Western blot analysis of various bovine tissues with human NMT peptide antibody indicated a common prominent immunoreactive band with an apparent molecular mass of 48.5-50 kDa in all tissues. Additional immunoreactive bands were observed in brain (84 and 50 kDa), lung (58 kDa), and skeletal muscle (58 kDa). Activity measurements demonstrated that brain contained the highest NMT activity followed by spleen, lung, kidney, heart, skeletal muscle, pancreas, and liver. It appears therefore that mRNA and protein expression do not correlate with NMT activity, suggesting the presence of regulators of the enzyme activity.
肉豆蔻酰辅酶A：蛋白质N-肉豆蔻酰转移酶（NMT）是一种必需的真核酶，它催化将肉豆蔻酸共翻译转移到许多功能各异的重要蛋白质的氨基末端甘氨酸残基上。我们已分离出编码牛脾脏NMT（sNMT）的全长cDNA。sNMT的1248 bp单一长开放阅读框编码一个416个氨基酸的蛋白质，预测分子量为46,686 Da。该蛋白质编码序列在大肠杆菌中表达，产生了具有功能活性的50 kDa NMT。缺失诱变表明，C末端对活性至关重要，而从N末端删除多达52个氨基酸不会影响功能。其中一个N末端缺失导致NMT活性提高了三倍。基因组Southern分析表明，用三种不同的限制酶消化后出现两条强杂交带，这表明牛基因组中可能存在NMT基因的两个拷贝。用牛sNMT cDNA探测从牛脑、心脏、脾脏、肺、肝脏、肾脏和骨骼肌制备的总细胞RNA的RNA印迹杂交分析显示有一条单一的1.7 kb mRNA。用人NMT肽抗体对各种牛组织进行的蛋白质印迹分析表明，所有组织中都有一条明显的免疫反应带，表观分子量为48.5 - 50 kDa。在脑（84和50 kDa）、肺（58 kDa）和骨骼肌（58 kDa）中还观察到其他免疫反应带。活性测量表明，脑中NMT活性最高，其次是脾脏、肺、肾脏、心脏、骨骼肌、胰腺和肝脏。因此，mRNA和蛋白质表达似乎与NMT活性不相关，这表明存在该酶活性的调节因子。
